Abnormalities of the human limb are one of the most common human birth defects, yet little is known as to the genetic or molecular changes underlying these defects. The developing mouse and chicken limb have provided excellent models to understand limb development and pattern formation in the vertebrate embryo. By defining the key molecular mechanisms by which the limb is patterned and bone formation is controlled, we will gain a better understanding of the possible causes of human limb defects. Many studies to date on these model organisms have explored the function of a relatively small set of patterning genes. However, there are many gaps in our knowledge as to what regulates these key players, what are their downstream targets and how is thr information translated into the formation of bones of correct number, shape and size. This proposal focuses on two different approaches to help to close these gaps in our knowledge. First, we will continue an unbiased forward genetic screen in the mouse initiated in the past granting period to identify new genes that regulate limb development, in particular those required for the formation of the correct number and size of skeletal elements. Second, we will use a novel live imaging system to follow the behaviors of cells as they undergo the early steps of cartilage formation. These approaches will allow us to elucidate where, when and how these novel genes function to regulate limb development and skeletal formation. In Aim 1 we will explore the genetics of polydactyly (extra digit formation) and syndactyly (digit and soft-tissue fusion and/or digit loss) and determine the molecular mechanisms by which novel genes regulate development of the hand and foot. In Aim 2 we will expand the mutagenesis screen to identify a set of new mutations that affect similar aspects of limb development to broaden our knowledge of the genes involved and to provide new models for understanding human limb defects. This will also allow for the testing of genetic links between different pathways that have not yet been realized. Finally, in Aim 3 we will use a novel live imaging system to begin to explore the cell dynamics of limb mesenchyme as it undergoes cartilage formation. This will provide unique insight into the critical events that occur as an undifferentiated mesenchyme cell undergoes the transition to a chondrogenic cell. We will test the hypothesis that cells along the A-P axis exhibit distinct cellular behaviors and that these behaviors are altered in new and classical mutants of polydactyly and syndactyly. Together, these studies will build a deeper foundation for understanding the key genetic, cellular and molecular events that regulate embryonic limb development and provide new animal models of limb development that will be useful for understanding the underlying causes of human birth defects.